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Microfluidic platform for efficient Nanodisc assembly, membrane protein incorporation, and purification
Lab on a Chip ( IF 6.1 ) Pub Date : 2017-07-26 00:00:00 , DOI: 10.1039/c7lc00601b
James H. Wade 1, 2, 3, 4 , Joshua D. Jones 1, 2, 3, 4 , Ivan L. Lenov 1, 2, 3, 4 , Colleen M. Riordan 1, 4, 5, 6 , Stephen G. Sligar 1, 2, 3, 4, 7 , Ryan C. Bailey 1, 1, 2, 3, 4
Affiliation  

The characterization of integral membrane proteins presents numerous analytical challenges on account of their poor activity under non-native conditions, limited solubility in aqueous solutions, and low expression in most cell culture systems. Nanodiscs are synthetic model membrane constructs that offer many advantages for studying membrane protein function by offering a native-like phospholipid bilayer environment. The successful incorporation of membrane proteins within Nanodiscs requires experimental optimization of conditions. Standard protocols for Nanodisc formation can require large amounts of time and input material, limiting the facile screening of formation conditions. Capitalizing on the miniaturization and efficient mass transport inherent to microfluidics, we have developed a microfluidic platform for efficient Nanodisc assembly and purification, and demonstrated the ability to incorporate functional membrane proteins into the resulting Nanodiscs. In addition to working with reduced sample volumes, this platform simplifies membrane protein incorporation from a multi-stage protocol requiring several hours or days into a single platform that outputs purified Nanodiscs in less than one hour. To demonstrate the utility of this platform, we incorporated Cytochrome P450 into Nanodiscs of variable size and lipid composition, and present spectroscopic evidence for the functional active site of the membrane protein. This platform is a promising new tool for membrane protein biology and biochemistry that enables tremendous versatility for optimizing the incorporation of membrane proteins using microfluidic gradients to screen across diverse formation conditions.

中文翻译:

微流体平台,用于高效的纳米光盘组装,膜蛋白掺入和纯化

整体膜蛋白的表征由于其在非天然条件下的活性差,在水溶液中的溶解度有限以及在大多数细胞培养系统中的低表达而提出了许多分析挑战。纳米盘是合成模型的膜构建体,通过提供类似天然的磷脂双层环境,为研究膜蛋白功能提供了许多优势。膜蛋白在纳盘中的成功整合需要条件的实验优化。用于形成纳米盘的标准协议可能需要大量时间和输入材料,从而限制了对形成条件的简便筛选。充分利用微流控技术固有的小型化和有效的大众运输,我们已经开发了一种用于高效Nanodisc组装和纯化的微流体平台,并展示了将功能性膜蛋白整合到所得Nanodiscs中的能力。除了减少样品量外,该平台还简化了膜蛋白的整合过程,该过程只需数小时或数天即可将多阶段方案整合到可在不到一小时的时间内输出纯化的纳米光盘的单一平台中。为了证明该平台的实用性,我们将细胞色素P450掺入了可变大小和脂质组成的纳米光盘中,并提供了膜蛋白功能性活性位点的光谱证据。
更新日期:2017-08-22
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